The separation method of the present invention was conceived during research concerning analytical methods suitable for use in fluidic or microfluidic chips or cartridges. To better understand the advantages of the present invention, such analytical methods are described in detail below
A common method of analyzing for a certain substance or analyte involves the use of a solid phase which will selectively bind to the target substance or analyte which typically is a biomarker. In some assays a solid phase may on its surface carry and display specific capturing molecules which will specifically bind the biomarker. In order to detect and quantify said biomarker, the solid phase-biomarker complex may also react with another set of biomarker specific binding molecules attached to one or more tracer substance(s) forming solid phase-biomarker-tracer complex(es). In other assays, such as competitive immunoassays, the biomarker in the sample will compete with a defined amount of biomarker carrying a tracer substance in the binding to the solid phase. There are numerous ways of arranging and using the involved specific binders and target analytes, including various types of solid phase materials and tracer substances.
In many analytical systems, the solid phase comprises a fixed embodiment such as the walls of cavities (microtiter plate wells or microchannels and microcavities) or fixed structures, such as pillars or porous membranes, on which the capturing molecules, for instance antibodies, being complementary to the biomarker, for instance an antigen, are attached. Lateral or transversal flow, of the sample containing the target biomarker, through porous membranes is a preferred solid phase concept when it comes to binding of the target biomarker. This is due to the fact that the surface to volume ratio is very large in these membranes allowing for a large excess of the capturing molecules, e.g. antibodies, and hence very efficient binding of the target biomarker. However, these membranes are difficult to wash particularly when the tracer is a nanoparticle or agglomerates thereof. This difficulty is caused by unspecific binding or entrapment of the tracer substance in pocket-like structures within the membrane.
In other analytical systems, the solid phase may advantageously be spherically shaped nano- or microsized particles made in polymeric materials exposing a large surface area.
The tracer can be any type of substance that may be detected and measured by either optical, chemical, electrical, magnetic, radioactive means or combinations thereof. Further, the tracer substance may also be formulated as or associated with a particle. Such particles frequently used and detected by optical means includes metal colloids (gold, silver, iron and others), quantum dots, polymer (latex) particles containing or carrying dyes or fluorochromes, polymer, silica or other particles carrying signal generating molecules including enzymes or inorganic crystals such as upconversion nanoparticles (UCNPs). The particles used as tracer substances or carriers are usually in the nanometer range typically between 2 nm to 200 nm, but larger particles up to 100 μm may be used in some settings. The biomarker specific molecules attached respectively to the solid phase and the tracer substances may for example be antibodies that will specifically bind to the target biomarker, which is then referred to as the antigen. Frequently used alternatives to antibodies includes nucleic acid probes, avidin/streptavidin, lectins and aptamers as well as any (bio)receptor that will recognize and specifically bind to defined molecular structures of the ligand (i.e. the analyte or part of the analyte). Actually, a major part of all proteins within nature interact more or less specifically with some ligand which may be a defined structure of a large molecule or small molecules. Usually, the more specific and the higher the affinity the binding is the more suited the receptor ligand system is for designing analytical assays. To quantify the solid phase-biomarker-tracer substance complex, (in the following also referred to as a quantifiable bead complex) the tracer substance must display certain properties allowing identification and measurement. Optical readout systems are often particularly convenient as the detector may be placed outside the assaying device. Properties of optical tracer substances include light absorption, light scattering as measured by transmittance or reflectance as well as light diffraction and luminescent phenomena like chemoluminescence, fluorescence, upconversion phosphorescence and others including combinations thereof. The phenomena are typically referred when measuring colors, luminescence such as fluorescence and phosphorescence, diffraction, plasmon effects and others.
In most heterogeneous types of analytical assays the target biomarkers are first allowed to react with the solid phase and tracer substance in excess. Then the tracer substance not specifically bound to the solid phase is removed by washing.
To have the target biomarker bind to the solid phase and the tracer substance, respectively, they will have to interact directly. With high affinity binders, the more often the reactants, i.e. the biomarker, solid phase and tracer substance, interact or collide the faster the binding reaction. Thus, to obtain a fast assay one should establish reaction conditions with very high local concentration of the high affinity and specific binding substances. To establish such conditions, the solid phase should expose a large surface area crowded with specific, high affinity receptor molecules. Similarly the tracer substance carrying the second set of specific and high affinity receptor molecules should be present in high concentrations. Suspensions of nano- and microparticles expose large surface to volume ratios, similar to porous structures such as porous membranes. Fluidic movements that will further facilitate collisions between the reactants involved should be applied. Such movements may be obtained by moderate heating and stirring, but more preferably by making the sample and the reagents, including the tracer substance in case of non-competitive assays, pass/flow through the solid phase. Liquids flowing through solid phase materials like porous membranes, micro-channels, micro-pillar structures or stacked particles are hence preferred in designing efficient binding assays.
The same consideration as discussed in the previous paragraph applies equally well in a competitive heterogeneous assay. In such assays the solid phase expose a large surface area crowded with bound and labeled biomarkers.
Spherical micro- and nanoparticles are often preferred as solid phase materials for several reasons:                Particles have a very large surface to volume ratio.        Particles are efficiently functionalized in batch and can hence be mixed to form homogenous suspensions.        Particle suspensions can be dispensed in aliquots that may be used directly in liquids form or be formulated as dried aliquots such as tablets or freeze dried spheres.        Particles may be loaded with or made from materials that add distinct features to the particles. This includes distinct optical features like light absorption, light scattering, light emittance (luminescence) and more, as well as magnetism, radioactivity, catalytic/enzymatic, electrochemical, and other measurable features.        Particles may be transported within microfluidic systems when in suspension.        Particles may be efficiently mixed with the sample in solution.        Particles in suspensions may be separated from the solution by gravity, centrifugation, filtration, magnetic force (magnetic particles) or electric force or combinations thereof.        Particles may sediment, stay in suspension or float depending upon their density relative to the medium in which they are dispersed.        Particles can be made monodisperse and completely spherical both being porous or with a smooth solid surface without pores.        Particles may be packed or stacked in a variety of containers including columns.        Particles may be opaque (colored) or substantially transparent allowing compatibility and use in a variety of optically based measuring systems.        When compact monodisperse particles are stacked onto a filter, like in columns, they may form a porous lattice structure with regular and defined spacing. Liquids may flow in a controlled and reproducible manner through such columns.        
The most efficient way to make an analyte, and optionally a tracer substance in case of non-competitive assays, interact with immobilized capturing molecules, attached to the surface of solid phase particles, is to pack the particles onto a filter or slit in the form of a column and allow a solution containing the analyte, and/or reagents such as a tracer substance, pass through the column of particles. This can be done sequentially and in repetitive steps or by applying a mixture of both the analyte(s) and tracer substance(s) and letting them pass in one step.
After the reaction or binding step(s), the solution is separated from the solid phase and the solid phase washed to remove remaining excess tracer substances, labeled unbound analyte (as in competitive assays) etc., to obtain a consistent and accurate analysis.
The use of bead columns to obtain an efficient interaction between the beads and various tracer substances and/or analytes is known from analytical methods using microfluidic chips.
US 2009/0104077A1 discloses a method for performing an ELISA-assay (Enzyme Linked ImmunoSorbent Assay) in a microfluidic chip. The disclosed microfluidic chip has a fluid circuit which comprises a column structure filled with beads acting as the solid phase. An ELISA-type reaction is performed on the beads in the column structure by first forming a bead—biomarker-enzyme-labeled antibody complex. The excess of enzyme-labeled antibody is then removed by letting a washing liquid flow through the column by application of a centrifugal force. An important feature of said method is the ability to circulate the same washing fluid multiple times through the column to obtain an improved washing step. After the cleaning or washing step, a color-generating substrate is applied to the enzyme-labeled complexes and the generated color may for instance be measured at the column structure. At least one end of the column structure ends in a restricted passage preventing the solid phase beads from passing out of the column.
WO 2011/081530 A1 discloses a processing cartridge (i.e. a fluidic or microfluidic chip) for analyzing a test sample, for instance a biological sample such as whole blood. The cartridge is adapted for use in a centrifuge analyzing instrument. The cartridge may comprise particular fluid circuit elements described as traps. Such an element is used to form a column of solid phase particles (beads), wherein the particles may be retained while a fluid is passed through the column. By appropriately changing the direction of an applied centrifugal force relative to the cartridge, a fluid, containing various reactants (e.g. biomarker and tracer substance) which react with the solid phase particles, is passed through the column repeatedly. The design of the traps avoids the use of a filter, or narrow fluid path, to obtain a column of particles or beads. Removal of excess reagents is obtained by repeated washings of the particles. The description of the traps and their use, as well as the concept of microfluidic chips having a fluid circuit through which circuit a sample comprising an analyte, and various optional reagents and solvents, may be moved by the use of centrifugal force, are hereby incorporated by reference.
In other assay systems ferromagnetic particles are used as the solid phase to facilitate the washing or separation steps. A magnet is then temporary used to pull the particles to one wall of a reaction container and withholding them during separation from the liquid. When the reaction container is moved away from the magnet, the particles are free to be re-suspended in solution. However, the magnetic particles are optically dense due to their content of ferromagnetic materials and they will hence significantly quench the optical readout. For this reason ferromagnetic particles are not suited for use in combination with tracer labels that are being bound to the particles through the assay.
In the technical field of sample analysis, such as analysis of biomarkers, both the use of a stationary and a mobile solid phase in assays are known. The present invention relates inter alia to the use of such assays in microfluidic chips (i.e. processing cartridges) having a fluid circuit through which circuit a sample comprising an analyte, and various optional reagents and solvents, may be moved by the use of centrifugal force. Such microfluidic chips are disclosed in for instance Schultz et al. Clin. Chem. 1985, 31, 1457, U.S. Pat. No. 488,763, and the above-mentioned patent applications US 2009/0104077 A1 and WO 2011/081530 A1.
In heterogeneous type analytical assays, efficient and reproducible separation (washing) of the excess unbound tracer substance from the tracer substance being bound to the solid phase is essential for reliable analytical results. Efficient washing is particularly important in high sensitivity analyses.
A solid phase having a porous structure, such as porous membranes, is more difficult to wash efficiently than solid phases having smooth surfaces like those available on well walls, micro-pillars or spherical particles. However, a solid phase comprising spherical particles (e.g. beads) closely packed into a column experiences some of the same difficulties as those encountered in porous membranes regarding efficient and reproducible washing. The close interaction of the beads in a packed column provides a temporary porous structure (i.e. due to voids formed in between the beads) within which unbound tracer substances (i.e. tracer substance not bonded to the solid phase) is easily captured in a non-specific manner. In the prior art, such columns are washed by passing a washing liquid through the column. However, even if such a washing is repeated multiple times (ref. the disclosure of US 2009/0104077A1), at least some of the unbound tracer substance will remain captured in the porous structure and subsequently have a negative impact on the reproducibility and sensitivity of the assay.
The present invention relates to a method of separating beads of different densities. When used in a heterogeneous analytical assay, the method may for instance provide for the possibility of analyzing multiple analytes from the same sample in a sequential or parallel manner.